At the core of the behavioral changes characteristic of alcoholism is the rearrangement of gene expression in the brain of an addicted individual. However, there exists a fundamental gap in our understanding of how alcohol consumption produces this rearrangement and leads to subsequent changes in behavior. Previous work in a mammalian system described how a particular microRNA (miRNA) acts as a key intermediary in the production of alcohol-induced changes in gene expression that produce functional-alcohol tolerance. The objective of this application is to combine the power of next generation sequencing with Drosophila genetics to identify all alcohol-sensitive miRNAs and to functionally test them for a role in producing an alcoholism-related behavior: functional ethanol tolerance (inducible ethanol resistance). This research goal stems from collaboration between two labs, one specializing in alcohol regulation of miRNA in the mammalian system, the other being an expert in fruit fly genetics and epigenetic mechanisms of alcohol actions. Based upon preliminary data and review of the literature, the central hypothesis is that even acute ethanol exposure causes long lasting changes in miRNA expression that lead to a rearrangement of the brain transcriptome. To test this hypothesis, miRNA-Seq and RNA-Seq will be performed on flies that have been briefly sedated with ethanol. Three different time points will be assessed in order to capture early and late changes in miRNA expression and their targets. Then, each ethanol-responsive miRNA will be functionally tested to determine if it contributes to functional ethanol tolerance (inducible-ethanol resistance). In flies, ethanol tolerance is a product only of neural adaptation to ethanol. Therefore, we will manipulate miRNA expression levels in the nervous system using the Gal4/UAS system and then determine its effect on functional ethanol tolerance and ethanol resistance. Achieving these goals will help identify and evaluate the role of miRNAs in the neural adaptations that underlie ethanol tolerance and will contribute to our longer-term goal to understand how ethanol consumption modulates gene expression to promote alcoholism.